TGF-β Trap Research Articles

Analyses of functions of an anti-PD-L1/TGFβR2 bispecific fusion protein (M7824).

M7824 (MSB0011359C) is a novel first-in-class bifunctional fusion protein consisting of a fully human IgG1 anti-PD-L1 monoclonal antibody (with structural similarities to avelumab) linked to the extracellular domain of two TGFβ receptor 2 (TGFβR2) molecules serving as a TGFβ Trap. Avelumab has demonstrated clinical activity in a range of human cancers and has been approved by the Food and Drug Administration for the therapy of Merkel cell and bladder carcinomas. Preclinical studies have shown this anti-PD-L1 is capable of mediating antibody-dependent cell-mediated cytotoxicity (ADCC). In the studies reported here, it is shown that M7824 is also capable of mediating ADCC of a wide range of human carcinoma cells in vitro, employing natural killer (NK) cells as effectors, albeit not as potent as anti-PD-L1 employing some tumor cells as targets. The addition of the IL-15 superagonist fusion protein complex ALT-803 enhanced the ADCC capacity of both anti-PD-L1 and M7824, and to levels that both agents now demonstrated similar levels of ADCC of tumor cells. TGFβ is a known immunosuppressive entity. Studies reported here show TGFβ1 induced reduction of several NK activation markers as well as reduction of endogenous NK lytic activity and NK-mediated ADCC of tumor cells. These phenomena could be reduced or mitigated, however, by M7824, but not by anti-PD-L1. M7824, but not anti-PD-L1, was also shown to reduce the immunosuppressive activity of regulatory T cells on human CD4+ T-cell proliferation. These studies thus demonstrate the dual functionalities of M7824 and provide the rationale for its further clinical development.

Abstract 611: Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGFβ1 (TGFβ TRAP)

Abstract Background: Evasion of antitumor immunity is widely regarded as a hallmark of cancer. Tumor cells can avoid immune destruction by upregulating the immune checkpoint molecule PD-L1, and antibodies targeting the PD-1/PD-L1 axis have demonstrated clinical efficacy in several tumor types. Antitumor immunity can also be suppressed by TGFβ, a pleiotropic cytokine that reduces innate and adaptive antitumor immune responses. Additionally, TGFβ impacts tumor cells by activating a differentiation program called epithelial-mesenchymal transition (EMT), in which tumor cells lose epithelial features and gain mesenchymal characteristics. The gain of mesenchymal traits is associated with metastasis, stemness, and resistance to multiple therapeutic modalities, including chemotherapy and radiation. The objective of this work is to investigate the therapeutic potential of M7824 in non-small cell lung cancer (NSCLC). M7824 is a first-in-class bi-functional immunotherapeutic agent designed to target two negative regulatory pathways in immunosuppression. It is a novel fusion protein comprised of the extracellular domain of human TGFβRII covalently linked to the C-terminus of the Fc domain of the fully human anti-PD-L1 IgG1 antibody avelumab. Methodology: In vitro assays were conducted using NSCLC cell lines treated with TGFβ1 alone or in combination with avelumab or M7824 to assess the ability of M7824 to revert features of tumor cell EMT, including molecular marker expression, cell proliferation, and response to various chemotherapies. The ability of M7824 to mediate antibody-directed cellular cytotoxicity (ADCC) of TGFβ1-treated cells in vitro was also assessed. Results: Treatment with TGFβ1 induced mesenchymal marker expression, reduced cell proliferation, and enhanced resistance to docetaxel, paclitaxel, and gemcitabine. Treatment with M7824, but not avelumab, was able to both prevent and revert these effects of TGFβ1. M7824 also exhibited the ability to mediate ADCC of TGFβ1-treated cells in vitro. Conclusions: M7824 effectively antagonizes TGFβ1-mediated tumor cell mesenchymalization, including resistance to chemotherapy. These results suggest that M7824 has the potential to relieve immunosuppression and block treatment-resistance induced by TGFβ1 in patients with NSCLC. Citation Format: Justin M. David, Jeffrey Schlom, Yan Lan, Claudia Palena. Reversion of mesenchymal features in NSCLC cells using M7824, a first-in-class bi-functional immunotherapeutic agent targeting PD-L1 and TGF&#9461 (TGFβ&#946 TRAP) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 611. doi:10.1158/1538-7445.AM2017-611

Abstract CT080: Pharmacokinetic and pharmacodynamic analysis of M7824, a dual anti-PD-L1 and TGFβ TRAP molecule, in the first-in-human phase I dose escalation study

Abstract Introduction: M7824 is a bifunctional molecule that blocks both PD-L1 and TGFβ1-3. In this first-in-human phase I dose escalation study, in addition to monitoring the pharmacokinetics of M7824, we aimed to confirm its mechanism of action, particularly against the TGFβ cytokines. Methods: Patients were treated with M7824 intravenously at 5 dose levels of 0.3, 1, 3, 10, and 20 mg/kg every two weeks. PK analyses were performed from samples up to day 85 (7th dose). PD-L1 target occupancy was measured in CD3+ PBMCs by flow cytometry from patient blood collected at pre-dose, Day 2 (D2), D15, and D43. Further, the blood levels of TGFβ1-3 and pro-inflammatory cytokines were measured at these time points with an additional time point at D8 using analytically validated Luminex bead- and ECLIA-based multiplex immunoassays. Results: Results indicated a half-life of 4-8 days for M7824, which increased with dose up to 3 mg/kg, and was subsequently dose independent at dose levels of > 3 mg/kg. M7824 PK exposure during the first cycle increased in an approximately dose-proportional manner between 3 to 20 mg/kg, without significant accumulation within the first 85 days of treatment. There was PD-L1 target occupancy plateaued at about 80% for dose levels 3-20 mg/kg which was maintained throughout the dosing interval. Furthermore, there was a small (1.7 fold on D2) but significant induction of IFNγ at 0.3-20 mg/kg (p=0.001, n=19). TGFβ1-3 blood levels were reduced by a minimum of 99%, 92%, and 91%, respectively, at all time points for dose levels 1-20 mg/kg. At the dose of 0.3 mg/kg, TGFβ1-3 levels were depleted at D2 and D8, but not at D15 with an average reduction of 63%, 42% and 67%, respectively. Moreover, there was a correlation between the drug PK levels and the TGFβ trapping. Thus, TGFβ1-3 trapping was achieved in the circulation at drug dose level of 1 mg/kg and above. Conclusion: This study demonstrates in vivo evidence for highly significant trapping (>90%) of circulating TGFβ molecules (β1, β2, β3) by the bifunctional agent M7824 at dose levels of 1 mg/kg and higher, in addition to its high degree of peripheral PD-L1 target occupancy (80%). Further studies to evaluate the PK/PD, safety, and efficacy profile of M7824 are ongoing. Citation Format: Liang Cao, Zhigang Kang, Linghui Li, Julius Strauss, Isabelle Dussault, James Gulley. Pharmacokinetic and pharmacodynamic analysis of M7824, a dual anti-PD-L1 and TGFβ TRAP molecule, in the first-in-human phase I dose escalation study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr CT080. doi:10.1158/1538-7445.AM2017-CT080

Abstract 2615: Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways

Abstract Dual targeting of the programmed death ligand 1 (PD-L1) and transforming growth factor β (TGFβ) pathways, 2 key mediators of tumor immune evasion, may provide synergistic antitumor activity. The novel bifunctional fusion protein M7824 (MSB0011359C) is a fully human IgG1 monoclonal antibody against PD-L1, fused via a glycine-serine linker at the CH3-C terminus to the extracellular domain of human TGFβ receptor II, which functions as a TGFβ “trap.” Thus, M7824 is designed to exert independent and complementary anti-immunosuppressive functions by simultaneously blocking the PD-L1 and TGFβ pathways. The purpose of this study was to evaluate M7824 preclinically. M7824 was efficacious in multiple syngeneic tumor models (EMT-6, MC38, PANC02, 4T1, CT26, and GL261). In orthotopic EMT-6 and intramuscular MC38 models, M7824 showed superior additive antitumor effects compared to anti-PD-L1 antibody or TGFβ trap monotherapy. M7824 also inhibited spontaneous metastases in EMT-6 and 4T1 models. Furthermore, M7824 extended survival and conferred long-term protective antitumor immunity, as demonstrated by protection of cured mice against tumor cell rechallenge. Mechanistically, the dual anti-immunosuppressive activity of M7824 in the MC38 model resulted in unique activation of both the adaptive and innate immune systems, including increased tumor infiltration, proliferation, and cytotoxicity of CD8+ T cells; increased prevalence of splenic tumor antigen-specific CD8+ T cells; increased CD8+ TEM infiltration and CD8+ TEM/naïve ratio; increased tumor-infiltrated natural killer (NK) cells; increased ratio of M1/M2 macrophages; and decreased infiltration of tumor-associated neutrophils and myeloid-derived suppressor cells. Similar immunophenotypic changes were observed in the EMT-6 model. Activation of both the adaptive and innate immune systems contributed to the antitumor activity of M7824 in the MC38 model, as depletion of CD8+ T cells and NK cells led to a substantial decrease in M7824-mediated tumor regression; CD4+ T cells and antibody-dependent cellular cytotoxicity, however, appeared dispensable. Toxicologic evaluation in cynomolgus monkeys indicated M7824 was well tolerated, with only minor hematologic effects observed. Although M7824 is a bifunctional molecule, its antitumor activity can be further improved by combination with standard-of-care therapies: in the MC38 model, combining M7824 with radiotherapy or chemotherapy resulted in more effective inhibition of tumor growth and an increased frequency of tumor antigen-specific IFN-γ-producing CD8+ T cells vs either monotherapy. Collectively, our preclinical data suggest that M7824 elicits potent and synergistic antitumor activity via the simultaneous blockade of the PD-L1 and TGFβ pathways. Phase 1 clinical trials with M7824 are under way in patients with solid tumors. Citation Format: Yan Lan, Dong Zhang, Chunxiao Xu, Bo Marelli, Jin Qi, Huakui Qi, Guozhong Qin, Xiaomei Xu, Hong Wang, Aroop Sircar, Beatrice Brunkhorst, Eric Austin, Laszlo Radvanyi, Kin-Ming Lo. Preclinical evaluation and mechanistic characterization of M7824 (MSB0011359C), a novel bifunctional fusion protein targeting the PD-L1 and TGFβ pathways [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 2615. doi:10.1158/1538-7445.AM2017-2615

Preliminary results from a phase 1 trial of M7824 (MSB0011359C), a bifunctional fusion protein targeting PD-L1 and TGF-β, in advanced solid tumors.

3006 Background: M7824 (MSB0011359C) is a novel bifunctional fusion protein comprised of a fully human IgG1 monoclonal antibody against programmed death ligand 1 (PD-L1) fused to the soluble extracellular domain of transforming growth factor-β (TGF-β) receptor II, which acts as a TGF-β trap. We report preliminary data from a phase 1 trial of M7824 in patients (pts) with advanced solid tumors. Methods: NCT02517398 is a phase 1, open-label, 3+3 dose-escalation study. Eligible pts receive M7824 at 1, 3, 10, or 20 mg/kg Q2W until confirmed progressive disease, unacceptable toxicity, or trial withdrawal; treatment beyond progression is generally allowable. The primary objective is to determine the safety and maximum tolerated dose of M7824; secondary objectives include pharmacokinetics (PK), immunogenicity, and best overall response per RECIST v1.1. Results: 16 heavily pretreated pts with ECOG performance status 0-1 have received M7824. Our PK data show a dose-linear increase in exposure starting at a dose of 3 mg/kg; furthermore, M7824 saturates peripheral PD-L1 and sequesters any released plasma TGF-β1, -β2, and -β3 throughout the dosing period in a dose-dependent manner. Grade 3 drug-related treatment-emergent adverse events (TEAEs) occurred in 3 pts (skin infection secondary to grade 2 bullous pemphigoid [BP], lipase increased, and colitis with associated anemia); there were no grade 4-5 drug-related TEAEs. BP and colitis responded well to steroids. Colitis and its secondary events of anemia and rectal hemorrhage (in a previously radiated area) were considered dose limiting in 1 pt. There was preliminary evidence of efficacy across all dose levels, including 1 ongoing confirmed complete response (cervical), 1 durable partial response (pancreatic), a 25% reduction in the sum of diameters of target lesions after 2 doses of M7824 (cervical), and 2 cases of prolonged stable disease (pancreatic; carcinoid). Conclusions: Preliminary data from this phase 1 dose-escalation study suggest that M7824 has a manageable safety profile in pts with heavily pretreated advanced solid tumors. Early signs of clinical efficacy warrant further study. Clinical trial information: NCT02517398.

Abstract 3741: Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma

Abstract Transforming growth factor beta (TGF-β) isoforms are the primary mediators for TGF-β signaling via TGF-β receptors and downstream phosphorylation/dephosphorylation cascade. TGF-β is associated with a wide range of biological processes in oncology, including tumor cell invasion, migration, angiogenesis, immunosuppression, as well as regulation of tumor stem cell properties. Mouse 4T1 mammary carcinoma cell line is a transplantable tumor cell line that is highly tumorigenic and invasive and, unlike most tumor models, can spontaneously metastasize from the primary tumor in the mammary gland to multiple distant sites including lymph nodes, blood, liver, lung, brain, and bone. Considering rather challenging preclinical evaluation of antitumor activity in tumor models, mouse 4T1 mammary carcinoma model has been widely used in the literature for evaluation of TGF-β antagonists. In this report, we describe the efficacy of ISTH0047 - a potent and selective TGF-β2 antisense oligonucleotide - in murine 4T1 primary tumors and lung metastasis following tumor cell injection into the mammary fat pad (orthotopic tumor model) of syngeneic Balb/c mice. Consistent with literature data generated with other classes of TGF-β antagonists (e.g., small-molecule kinase inhibitors, antibodies or TGF-β trap agents), although limited antitumor activity was demonstrated on primary tumor growth, marked and statistically significant decrease of lung metastasis number was observed upon subcutaneous administrations of ISTH0047. In addition, side by side comparison with murine surrogates of CTLA-4 or PD-1 antibodies indicated similar efficacy of all test items on lung metastasis in this model. Taken together, these encouraging results pave the way for in-depth preclinical evaluation of both ‘seed and soil’ theory and efficacy of combination regimen (immunomodulation) for better clinical outcome. Citation Format: Katja Wosikowski, Kathy Hasenbach, Jutta Petschenka, Diana Barea Roldan, Sebastian Kreiter, Ugur Sahin, Guillaume Serin, Julie-Orlane Redon, Marc Hillairet de Boisferon, Francis Bichat, Hanna Kohonen, Michel Janicot. Antimetastatic activity of ISTH0047, a potent and selective TGF-beta 2 antisense oligonucleotide, in syngeneic lung metastatic model of mouse 4T1 mammary carcinoma. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3741.

Expression of vasorin (Vasn) during embryonic development of the mouse

The murine vasorin (Vasn) gene, initially known as Slit-like 2, encodes a transmembrane protein that shares structural similarities with the eponymous Slit proteins. However, whether it also shares functional similarities with these large secreted proteins remains to be elucidated. Here, we report expression of Vasn during embryonic and fetal development of the mouse using whole-mount in situ hybridization (WISH) and histochemical detection of β-galactosidase expressed from a targeted VasnlacZ knock-in allele. Comparison of whole-mount staining patterns of both approaches showed identical expression domains, confirming that Vasn promoter-driven β-galactosidase expression faithfully reflects endogenous Vasn expression. Vasn is highly expressed in vascular smooth muscle cells (hence the name), a finding consistent with a previous report on its human homolog VASN, whose extracellular domain was shown to function as a TGF-β trap (Ikeda et al., 2004). Most striking, however, is Vasn’s prominent expression in the developing skeletal system, starting as early as the first mesenchymal condensations appear. Moreover, distinct expression domains outside the bones, e.g., in the developing kidneys and lungs, suggest further roles for this gene in the mouse. Recently, it was shown that mitochondria-localized Vasn protects cells from TNFα- and hypoxia-induced apoptosis, and partial deletion of the Vasn coding sequence leads to increased sensitivity of hepatocytes to TNFα-induced apoptosis (Choksi et al., 2011). By providing a first comprehensive analysis of the Vasn expression pattern during mouse embryonic development, our study will help to further elucidate its biological functions.

P.7.a.006 Alteration of EEG characteristics in epileptic children during the treatment with antiepileptic drugs (carbamazepine)

Localized radiotherapy (RT) induces an immunogenic antitumor response that is in part counterbalanced by activation of immune evasive and tissue remodeling processes, e.g., via upregulation of programmed cell death-ligand 1 (PD-L1) and transforming growth factor β (TGF-β). We report that a bifunctional fusion protein that simultaneously inhibits TGF-β and PD-L1, bintrafusp alfa (BA), effectively synergizes with radiotherapy, leading to superior survival in multiple therapy-resistant murine tumor models with poor immune infiltration. The BA + RT (BART) combination increases tumor-infiltrating leukocytes, reprograms the tumor microenvironment, and attenuates RT-induced fibrosis, leading to reconstitution of tumor immunity and regression of spontaneous lung metastases. Consistently, the beneficial effects of BART are in part reversed by depletion of cytotoxic CD8+ T cells. Intriguingly, targeting of the TGF-β trap to PD-L1+ endothelium and the M2/lipofibroblast-like cell compartment by BA attenuated late-stage RT-induced lung fibrosis. Together, the results suggest that the BART combination has the potential to eradicate therapy-resistant tumors while sparing normal tissue, further supporting its clinical translation.